Construction and identification of lentiviral vector encoding shRNA against Smad3

Objective To construct a lentiviral vector expressing small-hairpin RNA(shRNA) targeting Smad3 gene.Methods The targeting sequence of Smad3 gene which can be effectively silenced in RNA inference was confirmed in our previous study.The cDNA containing both sense and antisense Oligo DNA fragments of the targeting sequence was designed,synthesized and cloned into the pGCSIL-GFP vector.The obtained lentiviral vector containing Smad3 shRNA was confirmed by PCR and sequencing.293T cells were cotransfected with lentiviral vector GC-shSmad3,pHelper 1.0 and pHelper 2.0.The titer of virus was tested according to the expression level of GFP.Results PCR and DNA sequencing demonstrated that the constructed lentivirus vector GC-shSmad3 produced Smad3 shRNA.The titer of concentrated virus was 3×108 TU/ml.Conclusion The lentivirus RNAi vector targeting Smad3 is constructed successfully.